Biomedical subjects
Publications and source records attributed to W F Bennett.
We report two patients with bronchoesophageal fistulae resulting from benign causes. The first resulted from erosion of an acquired middle esophageal diverticulum. The second had the prolonged clinical course of a presumed congenital fistula. The importance of barium examinations in evaluating chronic respiratory complaints is emphasized.
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Forty-one knees from adult cadavera (twenty female and twenty-one male) were dissected to study the relationship between the longitudinal axis of the patella and the angles of insertion into it of the vastus lateralis and vastus lateralis obliquus muscles. The mean and variance in the angles of insertion of the vastus lateralis obliquus tendon were found to be significantly different between men and women (p less than 0.05 and p less than 0.01, respectively). Three distinct anatomical patterns in the insertion of the vastus lateralis obliquus muscle were delineated. The vastus lateralis muscle, particularly the vastus lateralis obliquus, creates an important lateral force-vector on the patella.
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Hemangioblastomas of the spinal cord are uncommon, accounting for 1.6% and 2.1% of all spinal cord tumors (1). Until recently these vascular lesions were best evaluated angiographically. Several cervical hemangioblastomas have been studied by computed tomography with intravenous contrast medium enhancement (2-4). This report illustrates the use of computed tomography with combined intravenous and intrathecal contrast medium enhancement, which permitted preoperative diagnosis and treatment planning of an intramedullary hemangioblastoma.
A characteristic pattern of altered lumbar spine endplate morphology has been frequently noted in patients being evaluated for unrelated indications. The "owl's eyes" configuration of intravertebral disc material on axial transverse computed tomographic sections easily distinguishes this normal variant from pathologic processes. Two representative cases are provided for illustration, one of which on coronal reformations demonstrated the normal "Cupid's bow" contour previously described on frontal plain radiographs.
A recent case of absence of a cervical pedicle in a traumatized patient is presented. An incorrect radiological interpretation of the patient's plain films resulted in inappropriate management. Further radiological investigation, including computed tomography, revealed the underlying abnormality, and prevented subsequent misadventure . A review of the literature on this interesting abnormality and a complete differential diagnosis are presented.
Human tissue-type plasminogen activator (t-PA), obtained by expression in mammalian cells of recombinant DNA coding for the entire sequence of t-PA (rt-PA), was compared with natural activator from melanoma cell culture (mt-PA). In an in vitro system, composed of [125I]fibrinogen-labeled plasma clot suspended in circulating human plasma, rt-PA and mt-PA caused a very similar dose-related degree of fibrinolysis without causing extensive fibrinolytic activation and fibrinogen breakdown in the surrounding plasma. Urokinase only induced fibrinolysis at a 5- to 10-fold higher concentration and in association with extensive fibrinogenolysis. Intravenous injection of mixtures of labeled (0.4 microCi/kg) and unlabeled (2000 I.U./kg) mt-PA or rt-PA resulted in a rapid but similar disappearance of activity from plasma (T1/2 of 3 min) and specific accumulation of tracer in the liver. In rabbits with experimental jugular vein thrombosis, rt-PA and mt-PA caused a very similar dose-dependent thrombolysis without causing substantial systemic activation of the fibrinolytic system and fibrinogenolysis. Urokinase induced significant thrombolysis only at a 10-fold higher dose and this was associated with systemic fibrinolytic activation. Infusion of 96,000 I.U./kg (approximately equal to 1 mg/kg) of mt-PA or rt-PA over 4 hr induced approximately 70% lysis, whereas a 10-fold higher dose of urokinase yielded 35 to 40% lysis. Two subfractions of rt-PA differing in the extent of glycosylation had very similar thrombolytic properties. It is concluded that the potentially more readily available rt-PA could constitute a specific, fibrin-selective thrombolytic agent.
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A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffin cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidly than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded approximately 1-2 x 10(8) chromaffin cells per adrenal medulla at a purity of 95% or higher. On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolayer) cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells in suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Very few neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as there was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significantly with time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.
Suspension cultures of purified bovine adrenal chromaffin cells incorporated 32P from exogenous 32Pi into a protein of approximately M4 = 60,000 (isolated by discontinuous, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis). Phosphorylated tyrosine hydroxylase, purified from chromaffin cell supernatants by immunoprecipitation, co-migrated with the Mr = 60,000 band. Tryptic fragments prepared fom either the Mr congruent to 60,000 band or the immunoprecipitated tyrosine hydroxylase band were analyzed after separation with two-dimensional electrophoresis/chromatography. Two distinct 32P-peptides were present in either sample. After a 2-3-min lag period. 32P incorporation into both peptides was relatively linear with time for at least 20 min. In the presence of calcium, exogenous acetylcholine (100 microM) increased 32P incorporation into both of the 32P-labeled tryptic peptides whereas 8-bromo-cAMP (1 mM) increased 32P incorporation into only one of the two. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and MnCl2 inhibited the acetylcholine-induced phosphorylation of both tryptic peptides. Thus, tyrosine hydroxylase is phosphorylated in situ at more than one site, and the phosphorylation of these sites is affected differently by acetylcholine and 8-bromo-cAMP. The data imply that kinase activity other than (or in addition to) cAMP-dependent protein kinase activity attends tyrosine hydroxylase in the intact chromaffin cells and that multiple kinase activities may be involved in the short term regulation of catecholamine biosynthesis by afferent activity.
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We have previously shown that brief periods of high frequency synaptic stimulation of the rat hippocampus influence the endogenous phosphorylation of a 40,000 Mr brain protein (Browning et al.). The results of the present study demonstrate that this brain phosphoprotein is enriched in a purified mitochondrial fraction and co-migrates with the alpha-subunit of pyruvate dehydrogenase in sodium dodecyl sulfate polyacrylamide gels. Comparisons of total and partial proteolytic fingerprints indicate that the two proteins are essentially identical. In addition, the phosphorylation of the 40,000 Mr brain protein is sensitive to both dichloroacetate and magnesium as has been reported for pyruvate dehydrogenase. Taken together these data provide persuasive evidence that the brain protein is the alpha-subunit of pyruvate dehydrogenase and thereby raise the possibility that even very short periods of synaptic activity influence an enzyme of particular importance to mitochondrial metabolism in brain.
Changes in the activity of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1, PDH], elicited by inhibition of the phosphorylation of its 40,000 Mr alpha-subunit, were compared with changes in pyruvate-supported calcium accumulation by rat brain mitochondria. Dichloroacetate (DCA) produces concentration-dependent inhibition of the phosphorylation of intramitochondrial PDH alpha-subunit, which is accompanied by stimulation of PDH activity and calcium accumulation. DCA did not affect succinate- or ATP-supported mitochondrial calcium accumulation. The concentration of DCA giving half-maximal inhibition of the phosphorylation was almost identical to that giving half-maximal stimulation of PDH activity and calcium accumulation. PDH activity and pyruvate-supported calcium accumulation showed similar dependence on pyruvate concentration with respective apparent affinities for pyruvate of 40 microM and 30 microM, and both activities exhibited positive cooperativity. DCA modified only the maximal activity of PDH or the maximal calcium DCA modified only the maximal activity of PDH or the maximal calcium accumulation without changing either the apparent affinities for pyruvate or calcium or the Hill coefficients. These data provide evidence that calcium accumulation by mitochondria is tightly linked to PDH activity and that changes in the phosphorylation of the PDH alpha-subunit can be reflected in changes in the calcium-buffering ability of mitochondria. This suggests a possible mechanism by which a variety of manipulations, such as repetitive synaptic stimulation, can alter the regulation of internal calcium levels.
The rate of rat platelet shape change increases sigmoidally with respect to thrombin concentration under conditions where free Ca++ and ADP are limited to prevent platelet aggregation. In addition, the rate of shape change due to thrombin is considerably enhanced when the platelets are first treated with concanavalin A (Con A), an agent which itself produces shape change. In the presence of both agents the rate is considerably greater than the sum of the rates due to Con A and thrombin separately. This suggests that shape change itself may trigger increased platelet sensitivity to thrombin. One possible mechanism through which this might occur is that shape change promotes binding of thrombin to its surface receptors. If so, then Con A-induced shape change might facilitate binding of 125I-thrombin. Initial binding studies using previously described methods showed that nonspecific trapping of 125I-thrombin, which is bound specifically to platelets during shape change, prevents accurate measurements of thrombin binding. We found, however, that a portion of 125I-thrombin which is bound specifically to platelets forms a stable complex with a 40,000 dalton platelet protein. This complex cannot be disrupted by boiling in SDS buffer containing 2-mercaptoethanol. Linkage of 125I-thrombin to this protein is specific for thrombin, since it can be competed for by an excess of unlabeled thrombin and because a similar complex does not form using 125I-trypsin. Concentrations of Con A that induce platelet shape change also markedly increase the amount of complex produced by a given thrombin concentration. In addition, colchicine, an inhibitor of Con A-induced platelet function, markedly inhibits formation of the 125I-thrombin-receptor complex. We suggest that the sigmoidal response to thrombin might be related to appearance of new thrombin receptors on the platelet surface.
Exposure to low temperature causes platelets to change shape in a manner similar to the shape change that precedes secretagogue-induced serotonin release. Previous studies have shown that two proteins, of approximately 20,000 and approximately 40,000 Mr, become phosphorylated before secretion. We have investigated whether low temperature can induce phosphorylation of these proteins and/or serotonin secretion. The data indicate that low-temperature-induced shape change has no requirement for extracellular calcium, whereas phosphorylation of the two proteins and subsequent serotonin release both have strong calcium requirements. Because cold treatment is thought to influence platelet shape through an effect on microtubules, the events in the shape change-release sequence would seem to be ordered as follows: microtubule disassembly leads to shape change leads to protein phosphorylation leads to secretion.
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